Transporters play a central role in synaptic transmission. They are responsible for removal of neurotransmitters from the synaptic cleft and their storage in synaptic vesicles. In this project we propose to obtain mechanistic information at the molecular level on 2 transporters. These are GLT-1, a plasma membrane (Na+K+)-coupled transporter of the neurotransmitter glutamate, and EmrE-a unique bacterial multidrug transporter that provides an experimental paradigm to study the vesicular H+-coupled neurotransmitter transporters.In recent years, the study of bacterial homologues has provided important information on the structure of ion channels and transporters. The availability of new high-resolution structures of transporters from several families mark a new and stimulating era in defining mechanisms in molecular detail. We will progress towards understanding mechanisms by using the available structural and biochemical information to (i) further explore the residues in the binding pocket and to modify specificities and affinities of various substrates; (ii) study the molecular determinants of ion binding and (iii) to explore the conformational transitions that occur upon ion and substrate binding. In addition to impacting on the central question of the structural basis of ion-coupled transporter function, our studies may provide important clues for the role of these transporters not only under normal physiological conditions, but also in disease.